tbuckley/cabot-search huggingface dataset:
Abstract EC.08: Novel Candidate Variant In The Gene Coding For Receptor Tyrosine-protein Kinase ErbB-2 Is Associated With Left Ventricular Outflow Tract Obstruction Defects In Human
Authors: Minna Ampuja, Sabina Selenius, Ilkka Paatero, Iftekhar Chowdhury, Jenna Villman, Martin Broberg, Amanda Ranta, Tiina Ojala, Markku Varjosalo, Riikka Kivelä, Emmi Helle
Journal: Circulation Research (2023)

Congenital heart defects (CHD) are the most common congenital malformations. Both genetic and environmental factors, such as blood flow during development, are known to contribute to disease development. The genetic predisposition is likely in most cases oligogenic, and currently, robust evidence for few predisposing genes for monogenic forms exist. Here we show evidence for a rare variant in the ERBB2 gene as potentially causal for CHD. ERBB2 is known to be essential for cardiac development in animal models, but so far human data are lacking. We studied a Finnish cohort of left ventricular outflow tract obstruction (LVOTO) patients and their families and identified a variant in ERBB2 (R569C) that segregated with disease in three unrelated families with multiple affected members. We performed functional studies, and show that the mutated receptor is functional, as NRG1 stimulation phosphorylates the receptor. However, a protein-protein interaction assay showed that the mutated receptor is mainly located in ER/mitochondria, whereas the wild type ERBB2 is located at the plasma membrane. This was confirmed by a flow cytometry analysis showing decreased receptor presence in the plasma membrane of patient derived cells. Zebrafish embryos expressing the R599C mutant allele demonstrated altered myocardium structure and reduced contractility compared to the wild-type allele or a mock control. Finally, we differentiated induced pluripotent stem cells (iPSCs) from a patient with the variant into cardiomyocytes. Single-cell RNA sequencing demonstrated reduced expression of genes related to heart development, muscle contraction and response to oxidative stress in patient cells compared to healthy controls. As endothelial cells (EC) are important during cardiac development, we also differentiated patient and healthy control iPSCs into ECs. Transcriptomic analysis showed significant differences in the expression of angiogenesis and vasculature development related genes in the patient cells. In summary, ERBB2 R599C variant segregates with CHD patients in three unrelated families with LVOTO defects. The variant influences location of the protein and causes compromised heart function in zebrafish embryos. We propose ERBB2 as a new candidate gene for CHD.
Abstract P3160: Myocarditis Treatments Within Our Sights: Targeting Inflammatory Conversations In The Heart Using Extracellular Vesicles
Authors: Danielle Beetler, Katelyn Bruno, Joy Wolfram, DeLisa Fairweather
Journal: Circulation Research (2023)

Viral myocarditis is an inflammatory cardiomyopathy with no pharmacologic agent currently able to quell the immune mechanisms that damage the heart in their inflammatory crossfire to quell infections, without risk of immunocompromise. Sex differences are well known in inflammatory and/or infectious diseases. Females display more efficient, anti-inflammatory responses to external threats like viruses, while males launch a pro-inflammatory defensive response. Myocarditis is a classic case of an infectious, inflammatory disease being more frequent and more severe in men, while pre-menopausal women are most protected. Extracellular vesicles are biocompatible, highly abundant, stable messaging carriers with no transformative risk, and hold great promise as therapeutics due to their functional cargo. As EVs are soluble mediators of the conversations between cells, we seek to redirect protective anti-inflammatory messaging from pre-menopausal women to male mice, using a highly translational model of murine myocarditis. We derive EVs from omental fat, repurposing this source to derive a purified, therapeutic component. Our results show robust immune system control, with EV treatments effectively attenuating pro-inflammatory inflammasome and complement markers, while leaving regulatory components like M2b macrophages and complement component CR1 unchanged. Treated mice are healthier during acute disease and demonstrate better heart function than control mice, despite being a strain susceptible to myocarditis and subsequent dilated cardiomyopathy. Tests using EVs from similarly aged men do not demonstrate this anti-inflammatory capacity. Proteomic and RNA-sequencing content comparisons of these EVs from these two donor groups reveal important proteins and regulatory micro RNAs. Our data suggest that EVs from fat may be a novel anti-inflammatory treatment for viral myocarditis, however individual donors show varying therapeutic capacity, with EV content heavily influenced by sex and age. This study not only addresses a major therapeutic gap in the myocarditis field, but also provides functional and -omic based comparisons of EVs from donors differing only by sex, to test if female cardioprotection from myocarditis is transferrable.
Abstract P3182: Promoting Mitophagy By Enhanced Fatty Acid Oxidation Improves Phenotypes Of Heart Failure With Preserved Ejection Fraction
Authors: Akira Yoshii, Timothy McMillen, Rong Tian
Journal: Circulation Research (2023)

Although metabolic remodeling is known to contribute to the development of heart failure with reduced ejection fraction (HFrEF), the role of energy metabolism in HF with preserved ejection fraction (HFpEF) is poorly understood. Here we assessed cardiac energy metabolism in both HFpEF and HFrEF mice by multi-nuclear NMR spectroscopy. HFpEF was induced by a 2-hit of high fat diet (HFD) and Nω-nitrol-arginine-methyl-ester (L-NAME, in drinking water) for 10 weeks. Mice fed with chow diet, HFD or L-NAME administration alone were used as controls. HFpEF phenotypes were assessed as the exercise intolerance, pulmonary congestion, high E/e’ by echocardiography and left shift of pressure-volume relationship obtained from isolated hearts. High energy phosphate content, assessed by 31P NMR spectroscopy of isolated perfused hearts, showed a lower phosphocreatine (PCr) content in HFpEF hearts while ATP level was unaltered compared to all the controls, resulting a reduced PCr/ATP ratio similar to that in HFrEF induced by TAC. Decreased respiratory function and increased ROS production were observed in mitochondria isolated from HFpEF hearts suggesting mitochondrial dysfunction. Cardiac substrate oxidation profile, assessed by 13C-isotopmer analysis, showed a high dependency on fatty acid oxidation (FAO) in HFpEF hearts, which is opposite of HFrEF but similar to that in HFD hearts. However, PCr/ATP ratio and mitochondrial function were sustained in the HFD hearts with the same duration of treatment. We found that mitophagy was activated in HFD heart but did not change in HFpEF hearts despite similar extent of obesity suggesting that HFpEF heart is maladaptive to the metabolic stress. Promoting mitophagy by TAT-Beclin1 treatment improved mitochondrial function in HFpEF hearts and partially rescued cardiac diastolic dysfunction and lung congestion. Furthermore, enhancing FAO in HFpEF hearts by deleting acetyl-CoA carboxylase 2 stimulated mitophagy and improved HFpEF phenotypes. In summary, our results show that mitochondrial dysfunction is an important pathogenic mechanism for cardiac dysfunction in HFpEF. Maladaptation to metabolic stress in HFpEF hearts impairs mitochondrial quality control which can be improved by stimulating FAO.
Abstract P1053: Circulating Mitochondrial DNA Level Is A Biomarker Of Acute Myocardial Infarction And Mediates Cardiac Ischemia-reperfusion Injury
Authors: Yuxin Chu, Yutao Hua, Jamelle Brown, Celeste Bell, Jin He, Lihao He, Yunxi Chen, Sumanth D Prabhu, Cheng Zhang, Scott Ballinger, Min Xie
Journal: Circulation Research (2023)

Introduction: Ischemia/reperfusion (I/R) injury occurs after reperfusion, causing significant myocardial damage. Circulating mitochondrial DNA (mtDNA) levels are elevated in acute myocardial infarction patients, and act as Damage Associated Molecular Patterns (mtDNA DAMP) that induce inflammation via immune processes, including the toll-like receptor 9 (TLR9) pathway. However, mtDNA DAMP levels have not been measured in ST-elevation myocardial infarction (STEMI) patients before and after percutaneous coronary intervention (PCI) and the role of mtDNA DAMPs in I/R injury remains unknown. Hypothesis: Pre- and post-PCI levels of mtDNA DAMPs serve as markers of STEMI related cardiac injury. Limiting mtDNA DAMP levels by increasing autophagy or blocking their downstream effects on TLR9 will decrease cardiac I/R injury. Methods: Troponin and mtDNA DAMP levels were measured pre- and 24 hours post- PCI in 55 STEMI patients (90%-100% blockage) and compared to 37 healthy controls. MtDNA DAMP levels in cell-free plasma were measured by real-time qPCR. Using a C57BL6J mouse model of I/R injury, Tat-Beclin (TB, an autophagy inducer), ODN2088 (a TLR9 inhibitor) or negative control were given at reperfusion and infarct size, cardiac function, and mtDNA DAMP levels were determined. Results: Pre- PCI mtDNA DAMP levels were increased ~200 folds (p&lt;0.0001) in STEMI patients compared to healthy controls; post- PCI mtDNA DAMP levels were significantly decreased in the majority of patients. In contrast, troponin T levels increased, suggesting that mtDNA DAMP levels are early markers of STEMI. Interestingly, a greater decrease in mtDNA DAMP levels was associated with shorter symptom-to-PCI time. In mice, both TB induction of autophagy and TLR9 blockage by ODN2088 decreased infarct size by ~50% and preserved systolic function. As expected, TB induction of autophagy decreased mtDNA DAMP levels, while ODN2088 had no impact on mtDNA DAMP levels. Conclusions: Circulating mtDNA DAMP level is an early marker of STEMI and may predict the success of early PCI. Moreover, limiting the downstream effects of mtDNA DAMPs via induction of autophagy or blocking their action on TLR9 ameliorates cardiac I/R injury, and thus may provide a viable therapeutic avenue for STEMI patients.
Abstract P3099: Pathogenic Innate Immune Responses Blunt The Activation Of Pro-reparative Fibroblasts After Myocardial Infarction
Authors: Van K Ninh, Justin Yu, Rahul Sehgal, Kevin King
Journal: Circulation Research (2023)

Acute myocardial infarction (MI) leads to the development of a borderzone (BZ) that separates the ischemic zone from the surviving myocardium. The BZ is the site in which injured cardiomyocytes interact with diverse stromal and infiltrating immune cells that determine the course of tissue repair of the infarcted heart. We have recently identified spatially distinct transcriptional niches of cellular subsets using capture-based spatial transcriptomics in mice subjected to permanent ligation of the left anterior descending artery. Interestingly, we observed focal patterns of innate immune responses localized to the BZ microenvironment and a concomitant lack of stromal responses. We thus hypothesize that pathogenic BZ innate immune responses antagonize protective stromal cell responses important in the evolution and remodeling of the BZ.We tested the effects of the pro-inflammatory type I interferon response in vitro using cultured L929 fibroblasts treated with 10 ng/μL of IFNβ1, 10 ng/μL TGFβ1, or both. Fibroblast transcriptional activation was determined by gene expression of Postn, Sparc, Bgn, Acta2, and Col1 α 1 by qPCR analysis. The potent IFNβ1 cytokine significantly inhibited TGFβ-mediated fibroblast activation compared to TGFβ1-treated cells (0.42±0.05 vs 1.0±0.15, p &lt; 0.005).Fibroblast wound contracture and matricellular proteins have been shown to be critically important and protective in acute MI. This process was functionally measured by seeding fibroblasts into a 3D collagen gel and treated with IFNβ1, TGFβ1, or both and quantifying the difference in gel area. Using this functional assessment of fibroblast function, we show that IFNβ inhibits TGFβ-stimulated contraction of a 3D collagen hydrogel by again attenuating TGFβ-mediated fibroblast function (0.25±0.1 vs.60±0.11, p &lt; 0.005). This IFNβ1-mediated contractile inhibition was blocked by treatment with a neutralizing antibody against the IFNβ1 receptor. Our data enables us to understand how a given cell state changes based on the cells’ neighborhood and how this relates to cardiac remodeling post-MI. Understanding the exact cellular and molecular mechanisms of wound healing processes during the acute inflammatory event will be key to developing novel therapeutics.
Abstract P3058: Myocardial Infarction Impairs T Lymphopoiesis In Mice
Authors: Yonggang Ma, Danielle X Shane, Daria Konovalova, Sarah Y Yuan
Journal: Circulation Research (2023)

T lymphocytes, especially CD4 + T cells, play beneficial roles in regulating cardiac repair after acute myocardial infarction (MI). Some patients with MI show lymphopenia, a reduction in blood lymphocyte count. More importantly, lymphopenia negatively correlates with patient outcome. We have previously shown that circulating lymphocyte trafficking to the bone marrow contributes to lymphopenia post-MI. However, whether MI affects T cell production remains uninvestigated. The aim of this study was to investigate whether MI impairs T lymphopoiesis, the process of T cell development. Three to six months old male C57BL/6J mice underwent MI surgery by permanent ligation of the left anterior descending coronary artery. Sham controls were subject to the same procedure with the exception of artery ligation. At 7 days post-MI, we quantified thymic mass, cellularity, developmental thymocytes, and hematopoietic stem/progenitor cells in the bone marrow and thymus using multiparameter flow cytometry. Thymocyte apoptosis was evaluated using an Annexin V assay kit. Compared to sham controls, MI mice at day 7 post-MI exhibited smaller thymus, lower cellularity, and less thymocytes at different developmental stages, including double negative CD4 - CD8 - , double positive CD4 + CD8 + , and single positive CD4 + thymocytes. Early thymic progenitors (ETP) that generate thymocytes in the thymus were also lower in MI mice than sham counterparts. Interestingly, common lymphoid progenitors (CLP) in the bone marrow, the ancestors of ETP, were not affected by MI. To determine the reason that MI mice show less thymocytes, we assessed thymocyte apoptosis. The data showed that thymocytes in MI mice exhibited higher apoptotic rate than sham controls. In conclusion, our study, for the first time, demonstrates that MI impairs T lymphopoiesis, and this impairment mainly occurs in the thymus, but not in the bone marrow. The underlying mechanisms are related to increased thymocyte apoptosis.
Abstract P1097: Foxk1 Is A Regulator Of Cardiomyocyte Proliferation And Heart Regeneration
Authors: Javier Sierra-Pagan, Thijs Larson, Satyabrata Das, Xiao Ma, Tamas Alexy, qinglu li, Patricia Stan, Wuming Gong, Jianyi J Zhang, Mary G Garry, Daniel J Garry
Journal: Circulation Research (2023)

While the adult mammalian heart has limited regenerative potential, the neonatal mouse heart has a remarkable regenerative capacity. Our goal focused on defining new factors and mechanisms that could enhance repair and regeneration in the adult mouse heart. FOXK1 is a transcription factor that regulates cell cycle kinetics, myogenic stem cell proliferation and skeletal muscle regeneration. During development, its expression is restricted to progenitors, somites and the heart. However, its role in neonatal heart regeneration is unknown and warrants investigation. Here, we describe a novel role for FOXK1 as a regulator of cardiomyocyte (CM) proliferation and regeneration. Using computational analysis of single nucleus RNAseq datasets of neonatal CMs, we determined that FOXK1 was expressed in CMs. qPCR analysis of P1 hearts from control or Foxk1 null mice demonstrated that the absence of FOXK1 led to the dysregulation of Ccne1 and p21 expression, suggesting that FOXK1 could play a role in cell cycle kinetics in the neonatal heart. Control or Foxk1 null mice were pulsed with EdU which demonstrated that the absence of FOXK1 led to a decrease in CM proliferation in vivo. We then engineered a transgenic mouse model that overexpressed FOXK1 using the muscle creatine kinase ( MCK-Foxk1 ) promoter and isolated and cultured control or MCK-Foxk1 CMs (72 hrs), and determined that overexpression of FOXK1 led to a significant increase in CM proliferation. Furthermore, EdU pulsing studies in adult control or MCK-Foxk1 mice demonstrated a significant increase in EdU incorporation in adult CMs of MCK-Foxk1 unperturbed mice compared to control. We then performed LAD ligation surgeries in control and MCK-Foxk1 adult mice. Echocardiography 2 months following LAD ligations showed a significant increase in both ejection fraction and fractional shortening in MCK-Foxk1 mice compared to controls. Single nucleus RNAseq analysis of control and MCK-Foxk1 adult hearts highlighted two distinct CM populations by UMAP analysis, with the latter demonstrating a higher expression of myoblast proliferative pathways. These results identify FOXK1 as a regulator of CM proliferation and heart regeneration by employing both knockout and transgenic mouse models.
Abstract P3062: Tet2 Maintains Genome Stablity In Cardiac Myeloid Cells
Authors: Yaling Dou, Yan Zhang, Tingting Hong, Shaohai Fang, James Martin, Yun Huang
Journal: Circulation Research (2023)

Myeloid cells play critical roles in the regulation of myocardial injury and repair. Clonal hematopoiesis (CH)-related mutations in genes, such as Ten-eleven Translocation 2 (TET2), can impair myeloid cells and are associated with increased risk of cardiovascular disease. To identify the subsets of myeloid cells that are impacted by Tet2 loss-of-function (LOF) and illuminate how Tet2 LOF disrupts normal myocardial repair, we analyzed the dynamics of myeloid cells in a myeloid-specific Tet2-deficient mouse model after ischemia-induced myocardial infarction (MI). We observed a substantial increase of neutrophils in the post-MI mice, which contributes to adverse outcomes during heart repair. Mechanistically, Tet2-deficient myeloid cells exhibited increased genome instability, accompanied with augmented activation of the intracellular innate immune response. Furthermore, pharmacological inhibiting intracellular innate immunity resulted in significantly reduced neutrophils in Tet2-deficient mice after MI and reversal of deleterious cardiac outcomes. Findings from this study are consistent with the conclusion that targeting the innate immune pathways is a viable therapeutic option to reduce the risk of cardiovascular diseases in CH individuals bearing TET2 LOF mutations.
Abstract P3161: Engineering Mesenchymal Stem Cell-derived Extracellular Vesicles To Enhance Their Therapeutic Efficacy In Cardiovascular Disease
Authors: Sarah Choudhury, Jennifer K Lang
Journal: Circulation Research (2023)

Background: Extracellular vesicles derived from cardiosphere-derived cells (CDC-EVs) have demonstrated the ability to regulate cardiomyocyte apoptosis and induce cardiomyocyte proliferation in pre-clinical models of acute MI. While CDC-EVs exhibit cardioprotective effects, they require invasive intramyocardial injections to achieve therapeutic efficacy, detracting from their translatability. Mesenchymal stem cells (MSC) also generate cardioprotective EVs (MSC-EVs), which have demonstrated therapeutic efficacy in models of cardiac disease following intravenous administration. However, MSC-EVs lack several of the cardioprotective miRs expressed by CDC-EVs, including miR146a, which has been shown to modulate cardiomyocyte apoptosis. The objective of our current study was to determine the effect of enriching MSC-EVs with cardioprotective CDC-EV miRNA on cardiomyocyte apoptosis. Methods: MSCs were transfected with miR-146a-5p mimics using Trans-IT TKO in serum free growth media. EVs were isolated using a combination of ultrafiltration and size exclusion chromatography (SEC) and characterized by size, morphology and expression of surface markers. RT-PCR was performed on EV samples to quantify miR-146a expression. Primary neonatal mouse cardiomyocytes were treated with MSC-EVs, miR-146a loaded MSC-EVs, CDC-EVs or matched volume vehicle control. Cardiomyocyte apoptosis was assessed by a TUNEL assay. Results: Treatment with miR-146a loaded MSC EVs reduced cardiomyocyte apoptosis compared with cardiomyocytes treated with unmodified MSC exosomes (14±1% vs. 31±3%) (mean ± SEM, p=0.001, one-way ANOVA). miR-146a loaded MSC-EVs and CDC-EVs demonstrated a similar level of reduction in cardiomyocyte apoptosis (14±1% vs. 15±1%). There was no difference in the level of apoptosis between cardiomyocytes treated with unmodified MSC exosomes and cardiomyocytes treated with vehicle control (31±3% vs. 27±1%) (mean ± SEM, p=0.366, one-way ANOVA). Conclusion: Engineering MSC-EVs to express miR-146a enhances their ability to reduce cardiomyocyte apoptosis in vitro . This highlights the potential of cargo engineering as a means to improve the therapeutic efficacy of EVs for cardiovascular disease.
Abstract P2082: Thrombosis In The Coronary Microvasculature May Be Responsible For Impaired Cardiac Relaxation And Diastolic Dysfunction
Authors: Rouault Paul, Sarah Guimbal, Lauriane Cornuault, Ninon Foussard, Célia Bourguignon, Virginie Grouthier, Frank Choveau, David Benoist, Alain-Pierre Gadeau, Thierry Couffinhal, Marie-Ange Renault
Journal: Circulation Research (2023)

Introduction: Heart Failure with Preserved Ejection Fraction (HFpEF) is proposed to be caused by endothelial dysfunction in cardiac small vessels. We previously identified Hhipl1,as a gene upregulated in the coronary vasculature of Leptin receptor deficient mice (Leprdb/db) a well-established mouse model of HFpEF. Importantly, Hhipl1 encodes for a decoy receptor of Desert Hedgehog (DHH) which is known to be critical for endothelial integrity. Objective: Our objective is to investigate the functional consequences of impaired Hedgehog (HH) signaling in the adult heart in order to identify novel mechanisms underlying the development of diastolic dysfunction. Method: Cdh5-Cre/ERT2, DhhFlox/Flox (DhhECKO) mice and their control littermates were administered with tamoxifen at 8 weeks of age to induce Dhh KO. Their cardiac function, exercise tolerance, and the phenotype of their coronary vasculature were assessed one month later. Results: DhhECKO mice presented significantly reduced exercise tolerance, increased end diastolic pressure (EDP) and Tau, with no change in their ejection fraction consistent with diastolic dysfunction. At molecular and cellular level, impaired cardiac relaxation in DhhECKO mice was associated with a significantly decreased phospholamban phosphorylation on Thr17 and an alteration of sarcomeric shortening in ex-vivo. Besides, as expected, DhhECKO mice exhibited phenotypic changes in their coronary vasculature including a prominent pro-thrombotic phenotype leading to an impaired capillary perfusion and local hypoxia. Notably, antiaggregant therapies (aspirin and clopidogrel) prevented the occurrence of both diastolic dysfunction and exercise intolerance in DhhECKO mice demonstrating for the first time that thrombosis may promote diastolic dysfunction. Importantly, we confirmed the critical role of thrombosis in Leprdb/db mice which also displayed increased cardiac small vessel thrombosis in comparison to control mice. Alike DhhECKO mice, we found that antiaggregants decreased EDP and improved exercise tolerance in Leprdb/db mice. Conclusion: Altogether, these results demonstrate that small vessel thrombosis may participate in the pathophysiology of heart failure with preserved ejection fraction.